Materials Polystyrene cuvettes (BSA -bovine serum albumin) Bradford reageni liqu
ID: 88046 • Letter: M
Question
Materials Polystyrene cuvettes (BSA -bovine serum albumin) Bradford reageni liquid protei Egg whites or milk other n unknown PBS (phosphate buffered saline) buffer 1. Preparation of unknown samples The unknown samples probably have too much protein and are out of the range of Beer s Law, These samples must be diluted, but by how much? Since the concentration is unknown, a dilution series will be assayed so that at least one sample is within the linear range of the standard curve. General you might want to prepare a sample that is 2x too high or 2x too low just in case. For this lab, dilute your samples 1:10, 1:100 and 1:1000. Be sure to label your tubes appropriately! 2. Place 20 ul of each diluted sample into clean, labeled tubes. 3. Development of the Standard Curve Using the chart below, you will prepare a series of samples of protein of known concentrations referred to as "standard proteins". Pipet 20 ul of each into a labeled tube. Calculate how much of each protein you added to each tube. Tube Standard mg of protein mg/ml 20 ul PBS 2 20 0.125 3 20 0250 20 0.500 20 ul 0.750 20 ul 1.00 20 ul 1.500 20 ul 2.000 1:10 unknown 1:100 unknown. 1:1000 unknown. 4 Add 1 ml Bradford Reagent to all tubes, 1-8 and the 20 kul ofeach diluted unknowns. Mix. The colorometric reaction should be complete after about 5 minutes, and the color should remain stable for about 1 hour, 5. for Using the Spectrophotometer warm up for at least 5 minutes before allow the machine to After turning on the Set the Wavelength: Set the wavelength using the nm tand buttons. making any TimpteExplanation / Answer
The amount of protein added is calculated by the formula,
Amount of protein (mg) = volume of protein standard (ml) x starting protein concentration.
The results are shown in the following table,
Volume of protein standard
microlitre
standard
mg/ml
TubeVolume of protein standard
microlitre
standard
mg/ml
mg of protein 2 20 0.125 0.0025 3 20 0.250 0.005 4 20 0.500 0.01 5 20 0.750 0.015 6 20 0.100 0.02 7 20 1.500 0.03 8 20 2.000 0.04